title: Stimulation of proglucagon gene expression by human GPR119 in enteroendocrine L-cell line GLUTag.
abstract: GPR119 is a G protein-coupled receptor expressed on enteroendocrine L-cells that synthesize and secrete the incretin hormone glucagon-like peptide-1 (GLP-1). Although GPR119 agonists stimulate L-cell GLP-1 secretion, there is uncertainty concerning whether GLP-1 biosynthesis is under the control of GPR119. Here we report that GPR119 is functionally coupled to increased proglucagon (PG) gene expression that constitutes an essential first step in GLP-1 biosynthesis. Using a mouse L-cell line (GLUTag) that expresses endogenous GPR119, we demonstrate that PG gene promoter activity is stimulated by GPR119 agonist AS1269574. Surprisingly, transfection of GLUTag cells with recombinant human GPR119 (hGPR119) results in a constitutive and apparently ligand-independent increase of PG gene promoter activity and PG mRNA content. These constitutive actions of hGPR119 are mediated by cAMP-dependent protein kinase (PKA) but not cAMP sensor Epac2. Thus, the constitutive action of hGPR119 to stimulate PG gene promoter activity is diminished by: 1) a dominant-negative Gαs protein, 2) a dominant-negative PKA regulatory subunit, and 3) a dominant-negative A-CREB. Interestingly, PG gene promoter activity is stimulated by 6-Bn-cAMP-AM, a cAMP analog that selectively activates α and β isoforms of type II, but not type I PKA regulatory subunits expressed in GLUTag cells. Finally, our analysis reveals that a specific inhibitor of Epac2 activation (ESI-05) fails to block the stimulatory action of 6-Bn-cAMP-AM at the PG gene promoter, nor is PG gene promoter activity stimulated by: 1) a constitutively active Epac2, or 2) cAMP analogs that selectively activate Epac proteins. Such findings are discussed within the context of ongoing controversies concerning the relative contributions of PKA and Epac2 to the control of PG gene expression.
authors.affiliation: Department of Medicine, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Oleg G
authors.initials: OG
authors.lastname: Chepurny
authors.forename: Daniela
authors.initials: D
authors.lastname: Bertinetti
authors.forename: Mandy
authors.initials: M
authors.lastname: Diskar
authors.forename: Colin A
authors.initials: CA
authors.lastname: Leech
authors.forename: Parisa
authors.initials: P
authors.lastname: Afshari
authors.forename: Tamara
authors.initials: T
authors.lastname: Tsalkova
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.forename: Hans-G
authors.initials: HG
authors.lastname: Genieser
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.forename: George G
authors.initials: GG
authors.lastname: Holz
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019858:Enteroendocrine Cells; D004983:Ethanolamines / Q000494:pharmacology; D005786:Gene Expression Regulation; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D054795:Incretins / Q000096:biosynthesis / Q000378:metabolism; D007739:L Cells; D051379:Mice; D052336:Proglucagon / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: C552740:2-(2-(4-bromophenyl)-6-methylpyrimidin-4-yl)aminoethanol; C508138:A-CREB protein; D004983:Ethanolamines; C519854:GPR119 protein, human; D020662:Guanine Nucleotide Exchange Factors; D054795:Incretins; D011743:Pyrimidines; D012333:RNA, Messenger; C486707:Rapgef4 protein, mouse; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D052336:Proglucagon; D052216:Glucagon-Like Peptide 1; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases